Breeding grasses for capacity to biofuel production or silage feeding value: an updated list of genes involved in maize secondary cell wall biosynthesis and assembly

In the near future, maize, sorghum, or switchgrass stovers and cereal straws will be a significant source of carbohydrates for sustainable biofuel production, in addition to the current use of grass silage in cattle feeding. However, cell wall properties, including the enzymatic degradability of structural polysaccharides in industrial fermenters or animal rumen, is greatly influenced by the embedding of cell wall carbohydrates in lignin matrix, and the linkages between lignins, p-hydroxycinnamic acids, and arabinoxylans. Breeding for higher and cheaper biofuel or silage production will thus be based on the discovery of genetic traits involved in each cell wall component biosynthesis and deposition in each lignified tissue. Due to its considerable genetic and genomic backgrounds, maize is the relevant model species for identifying traits underlying cell wall degradability variations in grasses. Maize genes involved or putatively involved in the biosynthesis of cell wall phenolic compounds, cell wall carbohydrates and regulation factors were therefore searched for using data available in grass, Arabidopsis, and woody species (mostly poplar and eucalyptus). All maize ortholog genes were searched for using protein sequences and a “blastp” strategy against data available in the www.maizesequence.org database. Genes were also mapped in silico considering their physical position in the same database. Finally, 409 candidate genes putatively involved in secondary cell wall biosynthesis and assembly were shown in the maize genome, out of which 130 were related to phenolic compound biosynthesis, 81 were related to cell wall carbohydrate biosynthesis, and 198 were involved in more or less known regulation mechanisms. Most probable candidate genes involved in regulation and assembly of secondary cell wall belonged to the MYB (45 genes) and NAC (38 genes) families, but also included zinc finger and HDZipIII encoding genes. While genes involved in ferulic acid cross-linkages with other cell wall components were little known, several families putatively involved in (arabino)-xylan chain biosynthesis and in feruloyl transfer were shown, including especially arabinosyl-CoA-acyltransferases, feruloyl-AX b-1,2-xylosyl transferases, and xylan-O-3-arabinosyl transferases. This candidate gene list, which focused on genes and orthologs known to be involved in cell wall component biosynthesis and regulation, cannot be considered as exhaustive. Other genes, whose role in cell wall lignification and deposition have not yet been defined, should very likely be added to the list of candidates required for secondary cell wall assembly. Genes encoding proteins of still unknown function should also be added to the list, as several of the latter are probably involved in lignified tissue biosynthesis and deposition

A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons

We describe a rapid, high-throughput method to scan for new RNA editing sites. This method is adapted from high-resolution melting (HRM) analysis of amplicons, a technique used in clinical research to detect mutations in genomes. The assay was validated by the discovery of six new editing sites in different chloroplast transcripts of Arabidopsis thaliana. A screen of a collection of mutants uncovered a mutant defective for editing of one of the newly discovered sites. We successfully adapted the technique to quantify editing of partially edited sites in different individuals or different tissues. This new method will be easily applicable to RNA from any organism and should greatly accelerate the study of the role of RNA editing in physiological processes as diverse as plant development or human health

Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

Background: The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. Results: Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 59-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. Conclusion: All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner

Plant RNA editing

International audienceIn plants, post-transcriptional modification of transcripts includes C-to-U, U-to-C and A-to-I editing. RNA editing in plants is essential, with many mutants impaired in editing of specific sites exhibiting strong deleterious phenotypes, even lethality. The majority of editing in plants occurs in mitochondrial and plastid transcripts, however, A-to-I editing also occurs in cytosolic tRNAs. Here we review recent findings concerning the cellular machineries involved in the different types of editing, recent analysis of the proposed functions for editing, and recent models for its appearance and retention in different plant lineages

Plant RNA editing

International audienceIn plants, post-transcriptional modification of transcripts includes C-to-U, U-to-C and A-to-I editing. RNA editing in plants is essential, with many mutants impaired in editing of specific sites exhibiting strong deleterious phenotypes, even lethality. The majority of editing in plants occurs in mitochondrial and plastid transcripts, however, A-to-I editing also occurs in cytosolic tRNAs. Here we review recent findings concerning the cellular machineries involved in the different types of editing, recent analysis of the proposed functions for editing, and recent models for its appearance and retention in different plant lineages

Developmental co-variation of RNA editing extent of plastid editing sites exhibiting similar cis-elements

In tobacco, 30 of 34 sites in chloroplast transcripts that undergo C‐to‐U RNA editing can be grouped into clusters of 2–5 sites based on sequence similarities immediately 5′ to the edited C. According to a previous transgenic analysis, overexpression of transcripts representing one cluster member results in reduction in editing of all cluster members, suggesting that members of an individual cluster share a trans‐factor that is present in limiting amounts. To compare leaves and roots, we quantified the editing extent at 34 sites in wild‐type tobacco and at three sites in spinach and Arabidopsis. We observed that transcripts of most NADH dehydrogenase subunits are edited inefficiently in roots. With few exceptions, members of the same editing site cluster co‐varied in editing extent in chloroplasts versus non‐green root plastids, with members of most clusters uniformly exhibiting either a high or low editing extent in roots. The start codon of the ndhD transcript must be created by editing, but the C target is edited inefficiently in roots, and no NDH‐D protein could be detected upon immunoblotting. Our data are consistent with the hypothesis that cluster‐specific trans‐factors exist and that some are less abundant in roots, limiting the editing extent of certain sites in root plastids

Cell wall deposition and metabolism in wheat grain

International audienceThere is an amazing diversity of cell wall composition and cell wall polysaccharide structure in wheat grain that is only partly explained by the complexity of plant tissues and cell type functions present in this organ. This review presents the state of the art on the structure and diversity of cell wall polymers in mature wheat grain and recent knowledge on cell wall metabolism with a focus on development-associated changes in wheat grain cell wall polysaccharides and genes controlling their biosynthesis. The diversity of polysaccharide structure observed in endosperm cell walls is tentatively explained on the basis of polysaccharide properties and cell type function. (c) 2012 Elsevier Ltd. All rights reserved

Ecotype Allelic Variation in C-to-U Editing Extent of a Mitochondrial Transcript Identifies RNA-Editing Quantitative Trait Loci in Arabidopsis

In higher plants, RNA editing is a posttranscriptional process that converts C to U in organelle mRNAs. Although RNA editing in mitochondria occurs much more frequently than in chloroplasts, editing of exogenously supplied RNA substrates in vitro and in organello has shown that editing in the two organelles shares some common features. In particular, the 20 nucleotides upstream of the editing site play an important role in specifying the C to be edited. Biochemical approaches have allowed the identification of features of cis-sequences necessary for RNA editing to occur, but have failed to identify any of the components of the mitochondrial editing machinery. In order to implement a genetic approach for identification of editing factors, we have identified a polymorphism in the editing efficiency of a mitochondrial site between two ecotypes of Arabidopsis (Arabidopsis thaliana), Columbia (Col) and Landsberg erecta (Ler). In rosette leaves, an editing site within the ccb206 mitochondrial gene is more highly edited in Col than in Ler. Depending on the development stage and tissue analyzed, the difference in editing extent varies between the two ecotypes; for example, in floral buds, editing extent does not differ. Single-point regression analysis of the editing efficiency in a sample of recombinant inbred lines derived from a cross between Col and Ler allowed the identification of two quantitative trait loci controlling this trait. A member of the pentatricopeptide repeat protein family that carries a putative mitochondrial transit sequence has been identified near a major quantitative trait locus on chromosome 4

Revisiting the cell wall of the wheat endosperm

National audienceThe wheat grain is an important source of food, animal feed and industrial raw material. The grain starchy endosperm is a storage tissue that corresponds approximately to the wheat flour. Cell walls only account for about 3% of the endosperm weight but they are prominent for wheat end-use quality (milling, bread-making) and as dietary fibre, they have a major impact on nutritional quality. The cell wall polysaccharides in the wheat endosperm consist in 70 % feruloylated arabinoxylans (AX) and 20% mixed-linked beta-glucans (MLG) which are deposited in the walls during the grain development. Early works have shown that in addition to these major polysaccharides, cellulose and mannans are also found in minor amounts in the dry grain endosperm. More recently callose and xyloglucans were detected transiently in the developing endosperm. No pectins were ever detected although pectins accumulate in the endosperm of the closely related Brachypodium distachyon. By proteomic analysis of Golgi-enriched fractions obtained from the endosperm of developing wheat we identified glycosyltransferases (GT) belonging to families implicated in the synthesis of xylans, MLG, mannans, xyloglucans and pectins. We therefore decided to further analyse the cell wall composition in the endosperm. Positive signals were obtained for mannans, xyloglucans and several pectin domains (homogalacturonans, rhamnogalacturonans, galactans and arabinans) using specific antibodies and after removing the major polysaccharides. Interestingly, some of the minor polysaccharides accumulate evenly in the whole endosperm while others specifically in specialized cells. The transfer cells in the crease region appeared strongly labelled for galactans, arabinans and xyloglucans. The endosperm cell wall is therefore more complex than once thought. We are generating transgenic wheat to silence several genes potentially involved in the synthesis of these polymers to evaluate their role in the developing grain

A role for RNA editing in the regulation of plastid-encoded polymerase activity

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